The DNA/RNA-binding protein MSY2 marks specific transcripts for cytoplasmic storage in mouse male germ cells

Abstract

During spermatogenesis, male germ cells temporally synthesize many proteins as they differentiate through meiosis and become spermatozoa. The germ cell Y-box protein, MSY2, constituting approximately 0.7% of total protein in male germ cells, binds to a consensus promoter element, and shows a general lack of RNA-binding specificity. Combining immunoprecipitation and suppressive subtractive hybridization, we identified populations of germ cell mRNAs that are not bound or bound by MSY2. The former population is enriched in cell growth and ubiquitously expressed mRNAs, whereas the latter population is enriched for stored or translationally delayed, male gamete-specific transcripts. Chromatin precipitation assays reveal that most of the MSY2 target mRNAs are transcribed from genes containing the Y-box DNA-binding motif in their promoters. In transgenic mice, mRNAs encoding exogenous GFP are directed or not directed into the MSY2-bound fraction by promoters containing or lacking the Y-box motif, respectively. We propose that MSY2 marks specific mRNAs in the nucleus for cytoplasmic storage, thereby linking transcription and mRNA storage/translational delay in meiotic and postmeiotic male germ cells of the mouse.

Fig. 1.

MSY2 is abundant in male germ cells. MSY2 was quantified in an extract from dissociated spermatogenic cells by using an affinity-purified antibody to MSY2. Enriched populations of dissociated germ cells were homogenized in RIPA buffer that solubilizes >85% of total MSY2. Recombinant MSY2 was used as a control calibration protein.

Fig. 2.

Sucrose gradient fractionation of testis RNP particles. The gradients were fractionated into 18 tubes and divided for protein and RNA analysis (T, top; B, bottom). (A) Immunoblot of MSY2. The open arrow indicates the position of MSY2. (B) Northern blot of P2 mRNA. Gradients were centrifuged for 3 h at 100,000 × g rpm to maximally sediment the nonpolysomal RNPs. As a result, most of the polysomes are near the bottom (tubes 12–18). A 60% sucrose cushion prevented their pelleting.

Fig. 3.

Clustering of mRNAs bound and nonbound to MSY2 identified with SSH. The numbers in parentheses represent the number of mRNAs identified in the category. Proteins with multiple functions or involved in several biological processes were counted in multiple annotation categories. Black bars, MSY2-bound mRNAs; white bars, MSY2-nonbound mRNAs.

Fig. 4.

ChIP products were analyzed by using quantitative real-time RT-PCR. Twenty-one-day-old CD1 mice were used to maximize ease of chromatin sonication after DNA-protein crosslinking. Identical results were obtained with sexually mature CD-1 mice. The promoter sequences from 16 genes expressed in testis were analyzed. The data from three independent assays represent the average of six determinations ± SEM and are presented as the fold difference of target genes from the DNA immunoprecipitated with anti-MSY2 compared with controls where no antibody was added (the control level for each promoter was set as a value of 1) after normalization with input DNA amplification. Assigning each sample a rank based on fold enrichment, differences between ranks were analyzed by using the Kruskal–Wallis test. The five genes marked with stars were significantly different from the others (P < 0.001).